This application is a national stage filing under 35 U.S.C. xc2xa7371 of International Application No. PCT/CH94/00242 filed on Dec. 23, 1994, which International Application was not published by the International Bureau in English.
1. Field of the Invention
The present invention relates to a process for potentiating the immunogenicity of synthetic, weakly immunogenic antigens. In what follows, synthetic, weakly immunogenic antigens are understood to be compounds having peptide or protein structures which are produced either chemically or by means of recombinant DNA technology and which, following parenteral administration in an aqueous solution or in the form of an aluminum adsorbate, only trigger an unimportant immunological response with very low antibody titers and lacking or only small T-cell proliferation. This group of antigens will hereinafter be called synthetic antigens for the sake of simplicity. By definition the immune response to the synthetic antigens herein described is therefore negligible if they are administered in an aqueous solution. Incomplete Freund""s adjuvant (IFA) is used as an experimental reference preparation. IFA is a water in oil (W/O) preparation which is known to stimulate the humoral as well as the cellular immune response. However, IFA can only be used for testing purposes because of strong, undesirable side effects. By the term vaccine, formulations will hereinafter be understood which contain, in addition to the antigen, substances which themselves perform the function of purely inactive ingredients or an immunopotentiating function or even a combination of both functions. Purely inactive ingredients are, for example, water for dissolving the antigen for the parenteral administration, anti-microbial, isotonicity adjusting and pH-stabilizing inactive ingredients. Often immunipotentiating substances are called adjuvants, comprising, for example, insoluble aluminum salts (-phosphates, -hydroxides), certain lipopdlysaccharides, muramyl peptides, trehalose compounds, several cytokines, such as interleukine 1, lipophilic block copolymers (poloxamers). However, the experimental reference preparation incomplete Freund""s adjuvant and several administration forms for vaccines, such as liposomes, emulsions, nano-capsules, also have adjuvant properties. These forms of administration not only cause the formation of an antigen deposit in vivo, but also have immune-stimulating properties.
2. Description of the Related Art
The development of new vaccines and the improvement of existing vaccine formulations has gained in importance and urgency in the past years (E. Eppstein et al., New Adjuvants for Vaccines Containing Purified Protein Antigens, Advances in Drug Delivery Review 4, 233-253, (1990)). The production of synthetic antigens as well as the development of suitable adjuvant formulations and forms of administration which increase the immunogenicity of weakly immunogenic compounds are in the forefront. The target of the development of new antigens is, on the one hand, diseases, such as AIDS, malaria, tuberculosis, cholera, hepatitis A, cancerous diseases, against which there are as yet no or only insufficiently active vaccines; on the other hand efforts are directed toward the replacement of the antigens contained in the traditional vaccines, such as inactivated viruses, bacteria or toxoids, by low-molecular peptides and proteins which are easier to produce and to purify and can be better characterized and which have the antigenic regions of the actual infectious agents in their structure. Such antigen peptides and proteins can be obtained at high purity biochemically or by means of recombinant DNA technology. This new generation of synthetic antigens has peptide sequences (epitopes) in its chemical structure which stimulate antigen-specific TH- (helper), TC- (cytotoxic) and B-lymphocytes. Hereby the so-called TH-, TC- and B-cell epitopes can each be individually present or can be covalently linked into a chimeral B-T epitope. Since these gene-technologically or chemically produced antigens have in general low molecular weights of approximately 500 to 2,000, their immunogenicity is very weak in contrast to toxoids of molecular weights of 50,000 to 150,000 or in contrast to particular antigens, such as inactivated viruses and other microorganisms.
Strategies for immunogenicity potentiation of synthetic antigens known up to now are based on increasing in a first step the molecular weight of these antigens by covalent linkage and, in a second step, on incorporating these higher molecular structures into immunopotentiating formulations.
It is known that an increase of the molecular weight can be achieved in that the synthetic antigen is covalently bound to high-molecular carrier proteins, such as diphtheria and tetanus toxoids, bovine serum albumin, keyhole limpet hemocyanin. These antigen carrier constructs, are disadvantageous because the employment of very expensive and relatively impure proteins from foreign organisms, because of the necessity for reactive and relatively toxic agents for the covalent linkage of the antigen and the carrier protein and because of the difficulty of purification as well as identity and purity testing of these compounds. On the other hand, it has also been proposed to increase the molecular weight of B-T epitopes by covalently linking them in a sort of branch structure to form multimers (J. P. Tam, Y.-A. Lu, Proceedings of the National Academy of Sciences of the USA 86, 9084-9088 (1989)). These constructs are called multiple antigen peptides, MAP in short.
It is further known that the combination of a B- and TH-epitope is essential for an antibody formation to take place, and that the combination of a TC-epitope with a TH-epitope can improve the cytotoxic lymphocyte response, also called CTL response, after administration in IFA (C. Widman et al., J. Immunol. Methods 155, 95-99 (1992)).
Various immunopotentiating formulations for such weak immunogen antigens and their higher-molecular constructs are described in PS-EP-A1-513,861. O/W emulsions containing immune stimulants are a primary part thereof. Their inherent thermodynamic instability, which are reflected by the appearance of coalescence during storage, is disadvantageous in connection with these coarsely dispersed or colloid-dispersed systems. In addition, the components of such liquid-dispersed formulations suffer from chemical changes, such as oxidation and hydrolysis. The described formulations furthermore mostly require immune stimulants, such as muramyl peptides, which are not quite harmless toxicologically. Finally, these formulations do not show any long term effects at all. To obtain protection by vaccination over several years it is therefore necessary to inject these vaccine formulations three to four times in accordance with a defined vaccination schedule (so-called xe2x80x9cboosterxe2x80x9d injections).
Furthermore, a system for the immune potentiation of antigens is known from the international publication WO 92/19263-A1, which employs so-called biodegradable microspheres, also called microcapsules or microparticles. It is disadvantageous with this method that the immune potentiation is mainly observed on the gastro-intestinal mucous membranes and therefore will probably show an effect only on relative few pathogens (so-called enterophatogenic microorganisms). The fact that the microparticles are administered in the duodenum and cannot be administered or taken perorally precludes a practical use, at least in humans. It furthermore appears in accordance with PS-EP-A2-333,523 that a balanced amount of fine (1 to 10 xcexcm) and coarser (20 to 50 xcexcm) microparticles seems to be an important prerequisite for the immune-potentiating effect. These requirements for the size range of the microparticles represent an additional effort during production and processing of the microparticles which appears to be disadvantageous.
It is the object of the invention to embed a synthetic antigen by means of specific biopolymers into biodegradable microparticles, to suspend these microparticles in a dispersion medium and to administer them parenterally, by means of which a potentiation of the systemic immunological response is caused.
In accordance with the invention this objective is attained by means of a process as set forth in the appended claims. Exemplary embodiments for this are described in Examples 1 to 6. The process of the invention will be explained in detail below by means of FIGS. 1 to 7.